Methods and kits using extended rhodamine dyes

Patent metadata

Field	Value
Publication number	US-9551029-B2
Application number	US-201514927908-A
Country	US
Kind code	B2
Filing date	Oct 30, 2015
Priority date	Jun 3, 1999
Publication date	Jan 24, 2017
Grant date	Jan 24, 2017

Abstract

Extended rhodamine compounds exhibiting favorable fluorescence characteristics having the structure

are disclosed. In addition, novel intermediates for synthesis of these dyes are disclosed, such intermediates having the structure

In addition, methods of making and using the dyes as fluorescent labels are disclosed.

First claim

What is claimed is:

1. A kit for fragment analysis comprising one or more nucleoside or nucleotide, a chain-terminating nucleotide analog and a primer, wherein one or more of the nucleoside or nucleotide is a labelled nucleic acid compound comprising the structure:

wherein B is a nucleobase;
W1 and W2 taken separately are selected from —H, —OH, and —F; and W3 is selected from —OH, monophosphate, diphosphate, triphosphate and phosphate analog
L is a linkage; wherein if B comprises a purine base, the linkage is attached to the 8-position of the purine, if B comprises a 7-deazapurine base, the linkage is attached to the 7-position of the 7-deazapurine, and if B comprises a pyrimidine base, the linkage is attached to the 5-position of the pyrimidine; and
D is an extended rhodamine dye comprising the structure:

wherein
R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 9 , R 10 , R 11 , and R 13 when taken alone are selected from —H, alkyl, alkyl independently substituted with one or more Z 1 , heteroalkyl, heteroalkyl independently substituted with one or more Z 1 , aryl, aryl independently substituted with one or more Z 1 , heteroaryl, heteroaryl independently substituted with one or more Z 1 , arylalkyl, arylalkyl independently substituted with one or more Z 1 , heteroarylalkyl, heteroarylalkyl independently substituted with one or more Z 1 , halogen, —OS(O) 2 OR, —S(O) 2 OR, —S(O) 2 R, —S(O) 2 NR, —S(O)R, —OP(O)O 2 RR, —P(O)O 2 RR, —C(O)OR, —NR 2 , —NR 3 , —NC(O)R, —C(O)R, —C(O)NR 2 , —CN, and —OR, wherein R is independently selected from —H, alkyl, heteroalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and linking group; or
R 1 taken together with R 2 , Y 1 , or Y 2 ; or
R 4 taken together with R 3 , Y 3 , or Y 4 ; or
R 5 taken together with R 6 , Y 3 , or Y 4 ; or
R 6 taken together with R 7 , Y 3 , or Y 4 ; or
R 10 taken together with R 9 or R 11 ; or
R 11 taken together with Y 1 , or Y 2 ; or
R 13 taken together with Y 3 or Y 4 are selected from alkyleno, alkyleno independently substituted with one or more Z 1 , heteroalkyleno, heteroalkyleno independently substituted with one or more Z 1 , aryleno, aryleno independently substituted with one or more Z 1 , heteroaryleno, and heteroaryleno independently substituted with one or more Z 1 ;
R 5 is selected from —H, alkyl, alkyl independently substituted with one or more Z 1 , heteroalkyl, heteroalkyl independently substituted with one or more Z 1 , aryl, aryl independently substituted with one or more Z 1 , heteroaryl, heteroaryl independently substituted with one or more Z 1 , arylalkyl, arylalkyl independently substituted with one or more Z 1 , heteroarylalkyl, and heteroarylalkyl independently substituted with one or more Z 1 ;
Y 1 , Y 2 , Y 3 , Y 4 when taken alone are selected from —H, alkyl, alkyl independently substituted with one or more Z 1 , heteroalkyl, heteroalkyl independently substituted with one or more Z 1 , aryl, aryl independently substituted with one or more Z 1 , heteroaryl, heteroaryl independently substituted with one or more Z 1 , arylalkyl, arylalkyl independently substituted with one or more Z 1 , heteroarylalkyl, and heteroarylalkyl independently substituted with one or more Z 1 ; or
Y 1 taken together with R1, R11 or Y 2 ; or
Y 2 taken together with R1, R11 or Y 1 ; or
Y 3 taken together with R 4 , R 5 , R 6 , R 13 or Y 4 ; or
Y 4 taken together with R 4 , R 5 , R 6 , R 13 or Y 3 are selected from alkyleno, alkyleno independently substituted with one or more Z 1 , heteroalkyleno, heteroalkyleno independently substituted with one or more Z 1 , aryleno, aryleno independently substituted with one or more Z 1 , heteroaryleno, and heteroaryleno independently substituted with one or more Z 1 ;
Z 1 is selected from —R, halogen, —OS(O) 2 OR, —SO 2 OR, —SO 2 R, —SO 2 NR, —S(O)R, —OP(O)O 2 RR, —P(O)O 2 RR, —CO 2 R, —NR 2 , —NR 3 , —NC(O)R, —C(O)R, —C(O)NR 2 , —CN, —O and —OR, wherein R is independently selected from —H, alkyl, heteroalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and linking group; and
wherein the linkage is attached to R 8 of the extended rhodamine dye.

2. A kit for fragment analysis comprising one or more nucleotide triphosphates, a chain-terminating nucleotide analog and a primer, wherein one or more of the nucleotide triphosphates is a labelled nucleic acid compound comprising the structure:

wherein B is a nucleobase;
L is a linkage; wherein if B comprises a purine base, the linkage is attached to the 8-position of the purine, if B comprises a 7-deazapurine base, the linkage is attached to the 7-position of the 7-deazapurine, and if B comprises a pyrimidine base, the linkage is attached to the 5-position of the pyrimidine; and
D is an extended rhodamine dye comprising the structure:

wherein
R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 9 , R 10 , R 11 , and R 13 when taken alone are selected from —H, alkyl, alkyl independently substituted with one or more Z 1 , heteroalkyl, heteroalkyl independently substituted with one or more Z 1 , aryl, aryl independently substituted with one or more Z 1 , heteroaryl, heteroaryl independently substituted with one or more Z 1 , arylalkyl, arylalkyl independently substituted with one or more Z 1 , heteroarylalkyl, heteroarylalkyl independently substituted with one or more Z 1 , halogen, —OS(O) 2 OR, —S(O) 2 OR, —S(O) 2 R, —S(O) 2 NR, —S(O)R, —OP(O)O 2 RR, —P(O)O 2 RR, —C(O)OR, —NR 2 , —NR 3 , —NC(O)R, —C(O)R, —C(O)NR 2 , —CN, and —OR, wherein R is independently selected from —H, alkyl, heteroalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and linking group; or
R 1 taken together with R 2 , Y 1 , or Y 2 ; or
R 4 taken together with R 3 , Y 3 , or Y 4 ; or
R 5 taken together with R 6 , Y 3 , Or Y 4 ; or
R 6 taken together with R 7 , Y 3 , Or Y 4 ; or
R 10 taken together with R 9 or R 11 ; or
R 11 taken together with Y 1 , or Y 2 ; or
R 13 taken together with Y 3 or Y 4 are selected from alkyleno, alkyleno independently substituted with one or more Z 1 , heteroalkyleno, heteroalkyleno independently substituted with one or more Z 1 , aryleno, aryleno independently substituted with one or more Z 1 , heteroaryleno, and heteroaryleno independently substituted with one or more Z 1 ;
R 5 is selected from —H, alkyl, alkyl independently substituted with one or more Z 1 , heteroalkyl, heteroalkyl independently substituted with one or more Z 1 , aryl, aryl independently substituted with one or more Z 1 , heteroaryl, heteroaryl independently substituted with one or more Z 1 , arylalkyl, arylalkyl independently substituted with one or more Z 1 , heteroarylalkyl, and heteroarylalkyl independently substituted with one or more Z 1 ;
Y 1 , Y 2 , Y 3 , Y 4 when taken alone are selected from —H, alkyl, alkyl independently substituted with one or more Z 1 , heteroalkyl, heteroalkyl independently substituted with one or more Z 1 , aryl, aryl independently substituted with one or more Z 1 , heteroaryl, heteroaryl independently substituted with one or more Z 1 , arylalkyl, arylalkyl independently substituted with one or more Z 1 , heteroarylalkyl, and heteroarylalkyl independently substituted with one or more Z 1 ; or
Y 1 taken together with R1, R11 or Y 2 ; or
Y 2 taken together with R1, R11 or Y 1 ; or
Y 3 taken together with R 4 ,

Assignees

• Applied Biosystems Llc

Inventors

• Lam Joe Y L
• Benson Scott C
• Menchen Steven M

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C07D209/60 Primary

  Naphtho [b] pyrroles; Hydrogenated naphtho [b] pyrroles · CPC title

• C07H21/00

  Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids · CPC title

• C09B11/24

  Phthaleins containing amino groups {; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes} · CPC title

• C07D471/06

  Peri-condensed systems · CPC title